Directed evolution of cytochrome P450DA hydroxylase activity for stereoselective biohydroxylation

Engineering of a hydroxylase for highly active and stereoselective biohydroxylation of C(sp(3))-H bonds attracts keen interest in synthetic chemistry. Herein, we report the development of a colorimetric high throughput screening assay for the directed evolution of cytochrome P450DA hydroxylase. The best triple-mutant P450DA-M3 (N190F/V356L/A486E) was identified with improvements in the turnover frequency (TOF) and total turnover number (TTN). P450DA-M3 exhibited good catalytic efficiency (up to 6750 TTNs) and stereoselectivity (up to 98% ee) in the biohydroxylation of diverse substrates. The heme domain structure of the P450DA was also solved for understanding the possible influence of these positive mutations.